The overall goal of this project is to elucidate various aspects of the molecular and cellular biology of type VII collagen, the major component of anchoring fibrils at the dermal-epidermal junction of human skin. Understanding the normal biology of type VII collagen is a critical prerequisite for further elucidation of the pathologic processes that affect anchoring fibrils in the dystrophic forms of EB. The importance of this consideration is emphasized by our recent demonstration of strong genetic linkage between the type VII collagen gene locus 9COL7A1) and the EB mutation in families with dominant dystrophic EB (1,2). Furthermore, type VII collagen synthesis and/or secretion has been shown to be abnormal in the skin of patients with dystrophic forms of EB, when examined by immunocytochemical techniques (3-5). Thus, type VII collagen is clearly a candidate gene in some families with dystrophic forms of EB. Towards elucidation of molecular and cellular biology of human type VII collagen, we propose the following specific aims: 1. ELUCIDATION OF THE COMPLETE cDNA SEQUENCE OF HUMAN TYPE VII COLLAGEN (a) Isolation and characterization of additional pro alpha 1 (VII) collagen cDNAs. (b) Comparison of amino acid sequences deduced from cloned cDNAs with peptide sequences in type VII collagen. (c) Devepment of polymerase chain reaction (PCR) primers which generate amplimers encompassing the entire mRNA of type VII collagen. (i) Direct sequencing of PCR products. (ii) Elucidation of PCR products by single strand conformation polymophism (SSCP) electrophoresis. 2. MAPPING OF BIOLOGICALLY IMPORTANT PROTEIN DOMAINS BY EXPRESSION OF FUSION PROTEINS (a) Elucidation of antigenic domains by epitope selection of antiboides. (b) Intermolecular interactions with other basement membrane components. 3. CHARACTERIZATION OF HUMAN TYPE VII COLLAGEN GENE (COL7A1) (a) Exon-intron organization and elucidation of splice junctions. (b) Identification of cis-regulatory elements in the 5' -flanking DNA. (c) Development of promoter/reporter gene constructs. 4. MODULATION OF TYPE VII COLLAGEN GENE EXPRESSION IN CULTURE (a) Assay of mRNA steady-state levels. (b) Transcriptional regulation in transient cell transfections. (c) Cytokine and pharmacologic modulation.